1. Field of the Invention
This present invention relates to modified antibodies. More specifically, the present invention relates to antibodies which have been modified by chemical conjugation with a heterobifunctional reagent and the use of these modified MAb's in the diagnosis and therapy of cancer and other mammalian disease.
2. Description of the Prior Art
The use of antibodies, particularly monoclonal antibodies ("MAb's"), has the potential to be an extremely valuable approach in the diagnosis and treatment of cancer. An important property of MAb's is their specificity for single antigens.
MAb's specific to tumor cell antigens have been produced. It has also been shown that MAb's may be efficiently coupled to adjuncts such as radionuclides. Such radio-labelled MAb's are useful in providing clinical data, such as tumor imaging from immunoscintography, also known as .gamma.-camera imaging or radioimmunoimaging. In immunoscintography, the MAb's are allowed to bind to the specific tissue or tumor types having the antigen recognized by the MAb's. The radionuclides are then visualized through the use of appropriate technology, such as through the use of a germanium camera. It is the unique specificity of MAb's which enables their application in immunoscintography of tumors and other types of tissues.
However, the use of MAb's in immunoscintography has been limited due to high background levels and low binding capacity of the MAb's to their antigens. Experimental studies suggest that the biodistribution of radio-labelled MAb's is dependent on many factors, including the specificity and clearance time of the antibody. For effective diagnosis of a tumor through immunoscintography, an antibody should be selected which binds to an antigen which is dense and homogeneous on the tumor cell surface. Effective diagnosis through immunoscintography also requires that the antibody chosen should effectively bind to the tumor antigen. However, often MAb's which bind to appropriate antigens do not offer the required high binding affinity. Additionally, even the use of those MAb's which bind with high affinity relative to other MAb's may still produce a high level of non-specific binding, resulting in high background levels when used in immunoscintography. Thus, there is a need for a method of improving the effectiveness of binding of MAb's in order to improve immunoscintography as a diagnostic tool.
Additionally, the cytotoxic effect of MAb's can be markedly increased by coupling to radionuclides, drugs or toxins. The unique specificity of MAb's has raised hopes of the development of immunotherapy. In immunotherapy, biologically active agents are delivered using MAB's to particular undesirable cell types, such as tumor cells, thereby affecting the undesirable cell types without affecting other cells of the subject. However, immunotherapies require extremely high specificity antibodies in order to avoid affecting healthy tissue. Thus, a method of increasing the specificity of MAb's would be highly beneficial in achieving the goal of a safe, effective immunotherapy.
Many MAb's remain in the circulation for several days following introduction into a subject. This is undesirable for at least two reasons. One reason is that circulating MAb's produces high background levels in immunoscintography. A second reason is that circulating MAb's coupled to radionuclides or other potentially cytotoxic agents may produce undesirable side effects in the subject after prolonged exposure. Thus, there is a need for a method of decreasing the clearance time of MAb's. Of course, too great a decrease would result in MAb's being eliminated before any effective use of the MAb's could be made. Thus, there is a particular need for a method of decreasing the clearance time of MAb's without substantially affecting uptake of MAb's by tumor or other target tissue.
One factor which is critical in determining both the specificity and clearance time of an antibody is the form of the antibody. As used herein, an "intact" antibody molecule will refer to an unmodified antibody molecule comprised of two heavy chains and two light chains. The intact, whole antibody molecule is seen on the reactant side of the chemical equation of FIG. 1. As seen in FIG. 1, the intact molecule is divided into the F.sub.c and the F.sub.ab domains. F(ab').sub.2, the bivalent form of the F.sub.ab fragment, may be produced through the digestion of the F.sub.c domain with a protease.
The two heavy chains (designated as "H" in FIG. 1) are held together by one or more disulfide bridges. In intact molecules these disulfide bridges are normally protected from reducing agents. It has been found however, that removal of the F.sub.c domain allows facile reduction of the disulfide bridges. Thus, F(ab'), the monovalent form, may be produced from F(ab').sub.2 through the action of a mild reducing agent. Parham, P., On the Fragmentation of Monoclonal IgG1, IgG2a, and IgG2b from BALB/c Mice, J. Immunol. 131: 2895 (1983), the disclosure of which is hereby incorporated by reference, describes a method for the production of F(ab') and F(ab').sub.2. A schematic representation of the changes believed to occur in this method is shown by the chemical equation of FIG. 1.
F.sub.c has been found to be responsible for much of the non-specific binding of antibody molecules. It is also believed that the molecular weight of the fragments is below the threshold for glomerular filtration, thus allowing for rapid elimination of the fragments. Therefore, one approach to increasing clearance time of antibodies for use in radioimaging has been to break down intact antibody into various fragments, such as Fab and its divalent form, F(ab').sub.2. As expected, these fragments are cleared from the body so rapidly that their utility is reduced. Moreover, these fragments may result in reduced uptake by tumor or other target tissue relative to intact antibody. Thus, the although the use of these fragments in immunoscintography may provide better clearance and a higher target tissue to background ratios than with intact MAb's, the absolute concentration of MAb's in the target tissue containing the antigen to which the MAb's will bind has been found to be up to as many as three times or more as much with intact MAb's as with either of the fragments.
Furthermore, both types of fragments are removed from the blood stream very rapidly. Accordingly, the time of effectiveness for diagnostic or therapeutic techniques using these fragments is very short.
Heterobifunctional reagents are reagents having two groups capable of participating in different reaction. For example, succinimidyl 3-(2-pyridyldithio)propionate (SPDP) is heterobifunctional in that its N-hydroxysuccinimide ester group reacts with amino groups and the 2-pyridyl disulphide structure reacts with aliphatic thiols.
Orlandi et al., Change in Binding Reactivity of an Anti-Tumor Monoclonal Antibody After the Introduction of 2-Pyridyl Disulphide Groups, Hybridoma 5:1-8 (1986), reported that an increase in the in vitro binding of MAb's raised against human ovarian carcinoma could be obtained after chemical conjugation with the heterobifunctional reagent, SPDP.
The conjugated MAb's used by Orlandi et al. had on average, 11 PDP groups per molecule. Orlandi et al. found that the modified MAb's increase their binding activity in vitro to an extent that molecules not detected by the unmodified MAb's can be detected. These researchers reported no studies of the use of the conjugated MAb's in vivo. Additionally, these researchers believed that molecules having a very low number of antigenic sites were detected by the conjugated MAb's. Accordingly, the PDP modified MAb's had greatly reduced target-cell specificity relative to the unmodified counterparts.
Thus, despite the above advances, there remains a need for modified antibody fragments exhibiting greater specific activity to tumor antigens, allowing more absolute concentration of antibody to accumulate in tumor, and also having relatively rapid clearance time from the blood pool, yet not so rapid to reduce therapeutic or diagnostic effectiveness.